In the United States alone, sepsis affects approximately 700,000 patients and leads to death of over 210,000 people per year. Sepsis is a clinical syndrome that results from a complex interaction between host and infectious agents, and is characterized by systemic activation of inflammatory and coagulation cascades. Hemodynamic changes, widespread microcirculatory disturbances and cellular alterations, leading to an uncoupling between blood flow and metabolic requirements, are implicated in the development of multiple organ dysfunction, responsible for most of deaths. Navigen believes that it has a unique means of preventing the hemodynamic changes and microcirculatory disturbances - the vascular leak - associated with sepsis. Our scientific co-founder, Dr. Dean Li, identified a novel receptor, Robo4, that is expressed in mature vessels and is upregulated following endothelial injury. Robo4, when activated by its protein ligand, Slit2, reduces vascular leak in vitro and in vivo. Our work has further illustrated that activation of the Robo4 receptor interferes with the downstream signaling cascades from multiple permeability factors including TNF-alpha, interleukins, and thrombin. We have cloned an active fragment of Slit2, Slit2N, which appears to have identical efficacy to Slit2, and is more easily produced than the native protein. Navigen has demonstrated that Slit2N is effective in reducing mortality in the animal model of sepsis induced by cecal ligation and puncture. Due to the challenges associated with protein therapeutics, Dr. Li sought to identify a small molecule that could affect the Robo4 pathway in a manner similar to Slit2N. Dr. Li and collaborators determined that a critical downstream step in Robo4 activation is the inhibition of a small GTPase, ADP ribosylation factor 6 (ARF6). Dr. Li and colleagues further determined that ARF6 can be blocked through inhibition of its ARF-GEF, cytohesin2/ARNO, using a small molecule inhibitor, NAV838. Dr. Li's laboratory, in collaboration with Navigen, has shown that inhibition of ARF6, either by blocking its ARF-GEF or through the inhibitory activation of Robo4 by Slit2N, produced similar phenotypes: inhibition of VEGF-induced migration in endothelial cells. Navigen is seeking funding under this application to confirm the efficacy of Slit2N in the treatment of sepsis and to determine whether NAV838, as a small molecule, may be an even more viable approach to treating sepsis than Slit2N. Navigen would anticipate advancing the superior compound forward to IND for eventual commercialization. The proposed research is intended to accomplish 3 goals: 1) develop animal models of sepsis induced by pseudomonas pneumonia (PAP) in which antibiotic therapy dose and timing are titrated to achieve seven-day mortality of approximately 50%, 2) test efficacy and indentify most efficacious dose of Slit2N combined with antibiotic therapy in models of CLP and PAP (PAP established in Aim 1), 3) determine whether NAV838 combined with antibiotics may have greater efficacy than Slit2N plus antibiotics by testing in animal models of CLP and PAP. PUBLIC HEALTH RELEVANCE: Sepsis is a clinical syndrome that results from a complex interaction between host and infectious agents, and is characterized by systemic activation of multiple inflammatory pathways, including cytokine network and coagulation. Mortality associated with severe sepsis ranges between 30 to 50 percent and sepsis is the leading cause of morbidity and mortality in surgical patients and trauma victims. Currently, there are no effective therapies for the treatment of Sepsis.